9. Vorlesung WS 2004/05Softwarewerkzeuge1 V9: Protein-Protein-Wechselwirkung - Consurf - Russell &...
-
Upload
ryan-larsen -
Category
Documents
-
view
214 -
download
0
Transcript of 9. Vorlesung WS 2004/05Softwarewerkzeuge1 V9: Protein-Protein-Wechselwirkung - Consurf - Russell &...
9. Vorlesung WS 2004/05
Softwarewerkzeuge 1
V9: Protein-Protein-Wechselwirkung
- Consurf
- Russell & Aloy Server
- Webseite mit Proteindomänen
- Beispiel zu Protein-Protein Wechselwirkung
Cytochrom c: Cytochrom c Oxidase
Barnase:Barstar
- Klausurvorbereitung
9. Vorlesung WS 2004/05
Softwarewerkzeuge 2
Consurf
http://consurf.tau.ac.il/
- degree of conservation at each amino acid site
is inversely related to its rate of evolution.-aim: identify functional regions on protein surface
by mapping degree of sequence conservation
within protein family-Use phylogenetic trees instead of MSA: by a
weighting scheme reduce influence of redundant
sequences
9. Vorlesung WS 2004/05
Softwarewerkzeuge 3
InterPreTS
http://www.russell.embl.de/interprets/
Protein-protein interactions are structurally
conserved for > 30% sequence identity.
Predict complexes of A:B based on
sequence homology to A‘ and B‘ when
structure of A‘:B‘ complex is available.
9. Vorlesung WS 2004/05
Softwarewerkzeuge 4
V9: Protein-Protein-Wechselwirkung
http://www.mshri.on.ca/pawson/research.html Pawson Lab
9. Vorlesung WS 2004/05
Softwarewerkzeuge 5
SH2 domains – universally used protein family
http://www.mshri.on.ca/pawson/research.html Pawson Lab
9. Vorlesung WS 2004/05
Softwarewerkzeuge 6
Proteins using SH2 domains
http://www.mshri.on.ca/pawson/research.html Pawson Lab
9. Vorlesung WS 2004/05
Softwarewerkzeuge 7
Binding properties of SH2 domains
http://www.mshri.on.ca/pawson/research.html Pawson Lab
9. Vorlesung WS 2004/05
Softwarewerkzeuge 8
Binding properties of SH2 domains
http://www.mshri.on.ca/pawson/research.html Pawson Lab
9. Vorlesung WS 2004/05
Softwarewerkzeuge 9
WW domain
http://www.mshri.on.ca/pawson/research.html
Pawson Lab
9. Vorlesung WS 2004/05
Softwarewerkzeuge 10
14-3-3 domain
http://www.mshri.on.ca/pawson/research.html
Pawson Lab
9. Vorlesung WS 2004/05
Softwarewerkzeuge 11
14-3-3 domain
http://www.mshri.on.ca/pawson/research.html
Pawson Lab
9. Vorlesung WS 2004/05
Softwarewerkzeuge 12
14-3-3 domain
http://www.mshri.on.ca/pawson/research.html
Pawson Lab
9. Vorlesung WS 2004/05
Softwarewerkzeuge 13
Ph
A
B
QHQ
B
2
ATPH + ADP + P+ 3- 2-
i
plasmamembrane
cytosol
lumen
lightharvestingcomplex
reactioncentre
cytochromeb f -complex
F F complex0 1
6H
+
light
cytochrome c
+2H
+2H 4H
4H+
+
Introduction: Photosynthesis
9. Vorlesung WS 2004/05
Softwarewerkzeuge 14
Docking strategy
No X-ray structure available for complex cyt c552:COX docking.
Flöck, Helms (2002)Proteins 47, 75
9. Vorlesung WS 2004/05
Softwarewerkzeuge 15
Protein-protein docking of cyt c552 and COX
Exercise on model system:
Complex of
yeast Cytochrome c Peroxidase
with iso-1-Cytochrome c
X-ray structure (Kraut et al. 1992)
Heme positions of crystal complex
and 19 best docked and
energy-minimized complexes.
Crystal complex has lowest energy.
Docked complex with second-best
energy has RMSD of only 2.0 Å.Flöck, Helms (2002)Proteins 47, 75
9. Vorlesung WS 2004/05
Softwarewerkzeuge 16
Protein-protein docking of cyt c552 and COX
Superposition of complexes
of Cyt c / Cyt c552 with
COX (bovine)
COX (P.d.)
Additonal loop of bovine COX
collides with c552 (grey).
Flöck, Helms (2002)Proteins 47, 75
9. Vorlesung WS 2004/05
Softwarewerkzeuge 17
Protein-protein docking of horse heart cyt c and COX
Best predicted complex of
horse heart cytochrome c with
cytochrome c oxidase from
Paracoccus denitrificans.
Almost identical with best
structure of Roberts et al. for
complex of horse heart
cytochrome c with bovine
cytochrome c oxidase. Docking
with DOT-program (1999).
Flöck, Helms (2002)Proteins 47, 75
9. Vorlesung WS 2004/05
Softwarewerkzeuge 18
Two favorable docking positions
Two favorable docking
positions for cyt c552
with COX.
The conformation of
the flexible linker and
of the N-terminal
helical anchor are
fictitous.
Flöck, Helms (2002)Proteins 47, 75
9. Vorlesung WS 2004/05
Softwarewerkzeuge 19
kinetic on-rates of protein-protein complexesfrom Brownian Dynamics simulations
McCammon group website (UCSD)
9. Vorlesung WS 2004/05
Softwarewerkzeuge 20
kinetic on-rates: exp data
Mutation kon 106 [M-1s-1] horse heart cyt c : COX
Wild type oxidase 3.7D135N 0.3N160D 2.8
Ionic strength [mM] P.d. cyt c552 : COX
10 4.135 1.5200 0.1
refs: Drousou, Malatesta, Ludwig, Eur J Biochem (2002) 269, 2980Maneg, Ludwig, Malatesta, J Biol Chem (2003) 278, 46734
9. Vorlesung WS 2004/05
Softwarewerkzeuge 21
brownian dynamics details
Electrostatics computed with programsUHBD (McCammon group)APBS (Baker, McCammon, Holst)
Atomistic brownian dynamics simulations with programSDA (Gabdoulline & Wade, 1997)
Simulation parameters:time step 2 ps – 10 pstranslat. diffusion constant 0.02 Å2 ps-1 rotational diffusion constant 4.0 10-5 radian2 ps-1 Radius b 115 ÅRadius c 540 Å
for each system 4 4000 runs
Flöck & Helms, Biophys.J.87, 65 (2004)
9. Vorlesung WS 2004/05
Softwarewerkzeuge 22
Iteration Algorithm
06 6
0 0 0 0
1 1
1/ ,N N
iji i ij i
j ji
Dy y t KT D t R D t
y
jF
1 11 3 1 3,..., , ,...,N NF F T TF
1 1ij ij
D
0
2i
i j ij
R
RR D t
Generalized force vector
Diffusion matrices
Random displacements 0, with:iR D t
Generalized coordinate vector
Dickinson, E., Allison, S.A. and McCammon, J.A. (1985) J. Chem. Soc. Farad. Trans. 2 81, 591
1 11 3 1 3,..., , ,...,N Ny r r
9. Vorlesung WS 2004/05
Softwarewerkzeuge 23
fulfil 1, 2, or 3 contact pairs among
D156:K79 D135:K86
A259:K73 D135:K86
S124:K86 Y122:G84
“naked” wild-type COX : horse heart cyt c 140mM
exp. rate with solubilizedCOX
Flöck & Helms, Biophys.J.87, 65 (2004)
9. Vorlesung WS 2004/05
Softwarewerkzeuge 24
Association of horse heart cyt c and „naked“ COX at 140mM.
Data for 3 reaction criteria.
Nice agreement with exp. trends!
Simulated mutation effects on on-rates
wild-type COX (3.7)
N160D (2.8)
D135N (0.3)
Flöck & Helms, Biophys.J.87, 65 (2004)
9. Vorlesung WS 2004/05
Softwarewerkzeuge 25
Simulated ionic strength effects on on-rates
Association of P.d. cyt c552 and „naked“ wild-type COX.
Data for 3 reaction criteria.
Nice agreement with exp. trends!
10 mM (4.1)
35 mM (1.5)
200 mM (0.1)
Flöck & Helms, Biophys.J.87, 65 (2004)
9. Vorlesung WS 2004/05
Softwarewerkzeuge 26
model of membrane environment
Flöck & Helms, Biophys.J.87, 65 (2004)
9. Vorlesung WS 2004/05
Softwarewerkzeuge 27
Start diffusion proteins only in
spherical cap above COX.
Inclusion of Membrane Environment
This scheme makes no difference for „naked“
COX:
comparison of original and spherical-cap
starting positions.
Flöck & Helms, Biophys.J.87, 65 (2004)
9. Vorlesung WS 2004/05
Softwarewerkzeuge 28
association rates with and without membrane environment for
horse heart cyt c : COX at 140 mM
Large effect!
Effect of membrane embedding for horse heart cyt c
„naked“ COX
with membrane potentials from APBS,
UHBD
Flöck & Helms, Biophys.J.87, 65 (2004)
9. Vorlesung WS 2004/05
Softwarewerkzeuge 29
association rates with and without membrane environment for
P.d. cyt c552 : COX at 140 mM
Association to „naked“ COX slower than for horse heart cyt c.
Small effect of membrane! Physiological relevance?
Effect of membrane embedding for P.d. cyt c552
„naked“ COX
with membrane
Flöck & Helms, Biophys.J.87, 65 (2004)
9. Vorlesung WS 2004/05
Softwarewerkzeuge 30
Computational studies: gain insight by switching isolated interactions off
all charges on
membrane charges off (cyt c552)
COX charges off (cyt c552)
membrane charges
off (cyt c)
COX charges off
(cyt c)
Flöck & Helms, Biophys.J.87, 65 (2004)
9. Vorlesung WS 2004/05
Softwarewerkzeuge 31
Protein – protein association
idealized reaction coordinate
G
free diffusion
protein B enters intoprotein A‘s zone of
electrostatic attraction directed diffusion
A and B form„encounter complex“
- electrostatically entangled,- no bound complex
to form complex,interfaces need tobe desolvated +
sidechains oriented
bound complex
9. Vorlesung WS 2004/05
Softwarewerkzeuge 32
Barnase:Barstar complex
extensively studied by Fersht group
barnase is an extracellular ribonuclease,
barstar its intracellular inhibitor
fast binding kon ~ 108 M-1s-1
high affinity kD ~ 10-14 M
binding stabilized by favorable electrostatic
binding energy (Wang et al. 2004)
association rates extensively studied by
Gabdoulline & Wade (1997)
Dong et al. (2003)
9. Vorlesung WS 2004/05
Softwarewerkzeuge 33
Aim of this study
Aim: clarify nature of encounter complex
Means: statistical analysis of brownian dynamics trajectories
9. Vorlesung WS 2004/05
Softwarewerkzeuge 34
Coordinate frame
Center of mass coordinates
of second protein
Rotational coordinates of
second protein
9. Vorlesung WS 2004/05
Softwarewerkzeuge 35
Different definitions of distance variable
For global view
For local view
9. Vorlesung WS 2004/05
Softwarewerkzeuge 36
how does the encounter state look like?
representative structures of the
encounter state ensemble
black balls: reaction atoms of
barstar in crystal structure
barnase
blue: cd1-2
red: cdmin
green: cdavg
purple: cdcenter
9. Vorlesung WS 2004/05
Softwarewerkzeuge 37
Zusammenfassung(1) Charakterisierung von Protein-Protein-Wechselwirkung heutzutage am
besten möglich per wissensbasierten Ansätzen (Sequenzhomologie).
(2) Energetische Charakterisierung noch schwierig
- Problem bei Protein-Protein Docking die beste Lösung zu finden
(3) Kinetik kann mittels Brownscher Dynamik charakterisiert werden.
Versuche, die 6-dimensionale G-Oberfläche durch Mapping der
Trajektorien zu erhalten.
So kann man den Encounter-Zustand als Minimum der freien Enthalpie
entlang einer geeigneten Reaktionskoordinate beschreiben
(4) Evolutionäre Relevanz: können verschiedene Teile der Proteinoberfläche für
verschiedene Phasen der Proteinassoziation verantwortlich sein?- Geladene Patches: langreichweitige Attraktion- Hydrophobe Patches: bilden Bindungsinterphase
Dann ergibt sich ein „evolutionärer Druck“ auf die ganze Oberfläche, nicht nur
auf das Bindungsinterface.